Plus-stranded (+)RNA viruses infect all kind of species and cause many serious diseases in humans, animals and plants, leading to major losses in food production and representing significant risk to human health and welfare. The least understood aspect of virus - host interaction is the contribution of the host cells. Due to their small genome sizes, RNA viruses greatly depend on the co-opted host proteins during replication. In spite of the significance of host proteins in viral pathogenesis, our current knowledge on host factors co- opted for virus replication is surprisingly incomplete. The relatively slow progress in this area is mainly due to the lack of tractable virus - host systems. Therefore, development of highly tractable model systems, such as Tomato bushy stunt tombusvirus (TBSV) and the yeast model host has promoted rapid and significant progress in our understanding of the roles of host factors in viral replication. The PI will use TBSV, which is currently one of the most advanced among viral systems to progress rapidly in understanding the mechanism of RNA replication and the role of the host proteins. Indeed, the PI's research, using a systems biology approach, including genome-wide screens and proteomics approaches, has led to a remarkably extensive data set containing close to 200 host proteins affecting tombusvirus replication, evolution and pathogenesis. Moreover, using novel cell-free system with a yeast extract capable of supporting authentic tombusvirus replication in combination with cell biology approach allows the PI to dissect the actual functions of the identified host proteins in tombusvirus replication. The PI has shown that the discoveries made in yeast are also relevant in the native host plant, thus validating the approach. These advances have put tombusviruses into the forefront of RNA viruses as a system of choice to further our understanding how RNA viruses "re-program" the infected cells. The PI's work will likely lead to major new insights into RNA virus replication and viral pathogenesis. Additional advantage of the similarity of TBSV replicase proteins to proteins of important pathogens, such as Hepatitis C virus (HCV), dengue virus, West Nile virus and other Flaviviruses and Pestiviruses. Collectively, the major advances with TBSV allow the PI to conduct experiments on the roles of host factors that are feasible with TBSV, and will stimulate development of new approaches for studying RNA replication and host - pathogen interactions for important human pathogens. This proposal focuses on a key host factor present in the viral replicase complex, namely GAPDH (glyceraldehyde-3-phosphate dehydrogenase) metabolic protein that binds to the viral RNA and essential for tombusvirus replication in yeast and in the native host plant. The proposed work will unravel the multiple functions of GAPDH in the viral replicase. These studies by the PI will certainly lead to further significant advances on the essential role of the host in RNA virus replication, evolution and virus-host interaction. Accordingly, GAPDH has been shown to bind to AU-rich sequences present in various RNA viruses, including hepatitis A virus (HAV), hepatitis C virus, and human parainfluenza virus type 3. The following are the major strengths of the proposal: (i) Viral RNA replication is clearly of immense importance for viruses to infect all types of living organisms. (ii) The combination of yeast and authentic cell-free assay developed by the PI is currently the most potent for studying the mechanism of host factors involvement in viral RNA replication. (iii) This research is expected to lead to groundbreaking new discoveries in viral RNA replication. The highly tractable in vitro and in vivo TBSV system developed by the PI could prove highly beneficial to studies of other, less amenable RNA viruses. The research holds promise of benefiting society by leading to groundbreaking results in the area of virus replication, host-virus interactions and the adaptation of viruses to their hosts. PUBLIC HEALTH RELEVANCE: RNA viruses, which pose significant risks to human health and cause major losses for agriculture, depend heavily on host factors to replicate in infected cells. The roles of the subverted host factors in virus replication are currently poorly understood due to the lack of tractable virus-host systems. The PI will use the powerful Tomato bushy stunt virus (TBSV)-yeast model system to dissect the role of a key co-opted host factor, namely GAPDH (glyceraldehyde-3-phosphate dehydrogenase) metabolic protein that binds to the viral RNA and essential for tombusvirus replication in yeast and in the native host plant. The proposed work will lead to deeper understanding of the role of the host in RNA virus infections and likely open new antiviral strategies.